High sequence fidelity in a non-enzymatic DNA autoligation reaction

The success of oligonucleotide ligation assays in probing specific sequence s of DNA arises in large part from high enzymatic selectivity against base mismatches at the ligation junction. We describe here a study of the effect of mismatches on a new nonenzymatic, reagent-free method for ligation of o ligonucleotides. In this approach, two oligonucleotides bound at adjacent s ites on a complementary strand undergo autoligation by displacement of a 5' -end iodide with a 3'-phosphorothioate group. The data show that this ligat ion proceeds somewhat more slowly than ligation by T4 ligase, but with subs tantial discrimination against single base mismatches both at either side o f the junction and a few nucleotides away within one of the oligonucleotide binding sites. Selectivities of >100-fold against a single mismatch are ob served in the latter case. Experiments at varied concentrations and tempera tures are carried out both with the autoligation of two adjacent linear oli gonucleotides and with intramolecular autoligation to yield circular 'padlo ck' DNAs. Application of optimized conditions to discrimination of an H-ras codon 12 point mutation is demonstrated with a single-stranded short DNA t arget.